logo
AAT Bioquest

ATTO 647N NHS ester

Product key features

  • Ex/Em: 645/663 nm
  • Extinction coefficient: 150,000 cm-1M-1
  • Reactive group: NHS ester
  • Easy Conjugation: Efficient labeling of primary amines on proteins and ligands, amine-modified oligonucleotides
  • High Quantum Yield & Stability: Provides bright fluorescence with high photostability, thermal resilience, and pH insensitivity across 2–11
  • Ozone-Resistant: Enhanced resistance to atmospheric ozone degradation ensures reliable performance in microarray applications

Product description

ATTO 647N is a rhodamine-based fluorescent dye optimized for use in the red spectral region, with similar spectral characteristics as Cy5. It is characterized by high molar absorptivity, a strong fluorescence quantum yield, and excellent thermal and photostability. The dye is moderately hydrophilic and exhibits optimal excitation within the 625-660 nm range, making it compatible with the 647 nm line of Krypton-Ion lasers and the 650 nm line of diode lasers. ATTO 647N maintains stable fluorescence across a broad pH range (pH 2-11), supporting its application under diverse experimental conditions. Upon conjugation to a substrate, the dye becomes cationic, carrying a net positive charge of +1. Unlike cyanine dyes, ATTO 647N demonstrates enhanced resistance to atmospheric ozone degradation, which increases its reliability in microarray applications. ATTO 647N is particularly effective for high-precision applications such as single-molecule detection, super-resolution microscopy techniques (e.g., SIM and STED), flow cytometry (FACS), fluorescence in situ hybridization (FISH), and various other biological assays.

The N-hydroxysuccinimidyl (NHS) ester of ATTO 647N is a widely used reagent for the conjugation of this dye to proteins or antibodies. NHS esters react selectively and efficiently with primary amines (such as the side chains of lysine residues or aminosilane-coated surfaces) at pH 7-9, forming stable covalent amide bonds. This property makes ATTO 647N NHS ester an excellent choice for labeling proteins, amine-modified oligonucleotides, and other amine-containing molecules.

Example protocol

PREPARATION OF STOCK SOLUTIONS

Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles

Protein Stock Solution (Solution A)
  1. Mix 100 µL of a reaction buffer (e.g., 1 M  sodium carbonate solution or 1 M phosphate buffer with pH ~9.0) with 900 µL of the target protein solution (e.g., antibody, protein concentration >2 mg/mL if possible) to give 1 mL protein labeling stock solution.

    Note: The pH of the protein solution (Solution A) should be 8.5 ± 0.5. If the pH of the protein solution is lower than 8.0, adjust the pH to the range of 8.0-9.0 using 1 M  sodium bicarbonate solution or 1 M pH 9.0 phosphate buffer.

    Note: The protein should be dissolved in 1X phosphate buffered saline (PBS), pH 7.2-7.4. If the protein is dissolved in Tris or glycine buffer, it must be dialyzed against 1X PBS, pH 7.2-7.4, to remove free amines or ammonium salts (such as ammonium sulfate and ammonium acetate) that are widely used for protein precipitation.

    Note: Impure antibodies or antibodies stabilized with bovine serum albumin (BSA) or gelatin will not be labeled well. The presence of sodium azide or thimerosal might also interfere with the conjugation reaction. Sodium azide or thimerosal can be removed by dialysis or spin column for optimal labeling results.

    Note: The conjugation efficiency is significantly reduced if the protein concentration is less than 2 mg/mL. The final protein concentration range of 2-10 mg/mL is recommended for optimal labeling efficiency.

ATTO 647N NHS ester Stock Solution (Solution B)
  1. Add anhydrous DMSO into the vial of ATTO 647N NHS ester to make a 10 mM stock solution. Mix well by pipetting or vortex.

    Note: Prepare the dye stock solution (Solution B) before starting the conjugation. Use promptly. Extended storage of the dye stock solution may reduce the dye activity. Solution B can be stored in the freezer for two weeks when kept from light and moisture. Avoid freeze-thaw cycles.

SAMPLE EXPERIMENTAL PROTOCOL

This labeling protocol was developed for the conjugate of Goat anti-mouse IgG with ATTO 647N NHS ester. You might need further optimization for your particular proteins.

Note: Each protein requires a distinct dye/protein ratio, which also depends on the properties of dyes. Over-labeling of a protein could detrimentally affect its binding affinity, while the protein conjugates of low dye/protein ratio give reduced sensitivity.

Run the Conjugation Reaction
  1. Use a 10:1 molar ratio of Solution B (dye)/Solution A (protein) as the starting point:  Add 5 µL of the dye stock solution (Solution B, assuming the dye stock solution is 10 mM) into the vial of the protein solution (95 µL of Solution A) with effective shaking. The concentration of the protein is ~0.05 mM assuming the protein concentration is 10 mg/mL, and the molecular weight of the protein is ~200KD.

    Note: We recommend using a 10:1 molar ratio of Solution B (dye)/Solution A (protein). If it is too less or too high, determine the optimal dye/protein ratio at 5:1, 15:1, and 20:1, respectively.

  2. Continue to rotate or shake the reaction mixture at room temperature for 30-60 minutes.

Purify the Conjugate

The following protocol is an example of dye-protein conjugate purification by using a Sephadex G-25 column.

  1. Prepare the Sephadex G-25 column according to the manufacturer's instructions.

  2. Load the reaction mixture (From "Run conjugation reaction") to the top of the Sephadex G-25 column.

  3. Add PBS (pH 7.2-7.4) as soon as the sample runs just below the top resin surface.

  4. Add more PBS (pH 7.2-7.4) to the desired sample to complete the column purification. Combine the fractions that contain the desired dye-protein conjugate.

    Note: For immediate use, the dye-protein conjugate must be diluted with staining buffer, and aliquoted for multiple uses.

    Note: For longer-term storage, the dye-protein conjugate solution needs to be concentrated or freeze-dried.

Characterize the Desired Dye-Protein Conjugate

The Degree of Substitution (DOS) is the most important factor for characterizing dye-labeled protein. Proteins of lower DOS usually have weaker fluorescence intensity, but proteins of higher DOS (e.g., DOS > 6) tend to have reduced fluorescence too. The optimal DOS for most antibodies is recommended between 2 and 10, depending on the properties of dye and protein. For effective labeling, the degree of substitution should be controlled to have 6-8 moles of ATTO 647N NHS ester to one mole of antibody. The following steps are used to determine the DOS of ATTO 647N NHS ester-labeled proteins.

Measure Absorption

To measure the absorption spectrum of a dye-protein conjugate, it is recommended to keep the sample concentration in the range of 1-10 µM depending on the extinction coefficient of the dye.

Read OD (absorbance) at 280 nm and dye maximum absorption (ƛmax = 663 nm for ATTO 647N NHS ester)

For most spectrophotometers, the sample (from the column fractions) needs to be diluted with de-ionized water so that the O.D. values are in the range of 0.1 to 0.9. The O.D. (absorbance) at 280 nm is the maximum absorption of protein, while 663 nm is the maximum absorption of ATTO 647N NHS ester. To obtain accurate DOS, ensure the conjugate is free of the non-conjugated dye.

Calculate DOS

You can calculate the DOS using our tool by following this link:

https://www.aatbio.com/tools/degree-of-labeling-calculator 

Spectrum

Product family

NameExcitation (nm)Emission (nm)Extinction coefficient (cm -1 M -1)Quantum yieldCorrection Factor (260 nm)Correction Factor (280 nm)
ATTO 488 NHS ester499520900000.800.220.09
ATTO 532 NHS ester5315521150000.900.220.11
ATTO 647 NHS ester6466661200000.200.080.04
ATTO 594 NHS ester6026211200000.850.260.51
ATTO 514 NHS ester510531115,0000.850.210.08
ATTO 565 NHS ester5625891200000.900.270.12
ATTO 390 NHS ester39047524000.900.460.09
ATTO 425 NHS ester438484450000.900.190.17
ATTO 495 NHS ester497525800000.20.450.37
ATTO 550 NHS ester5535741200000.800.230.10
ATTO 590 NHS ester5926211200000.800.390.43
ATTO 610 NHS ester6156321500000.700.030.06
ATTO 620 NHS ester61964112000010.510.040.06
ATTO 633 NHS ester6296511300000.6410.040.05
ATTO 655 NHS ester6616791250000.310.240.08
ATTO 680 NHS ester6796961250000.300.300.17
ATTO 700 NHS ester6997151200000.250.260.41
Show More (8)

Citations

View all 21 citations: Citation Explorer
Single-molecule enzymatic reaction dynamics and mechanisms of GPX3 and TRXh9 from Arabidopsis thaliana
Authors: Kuang, Yanmin and Guo, Xing and Guo, Aiyu and Ran, Xia and He, Yulu and Zhang, Yu and Guo, Lijun
Journal: Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2020): 118778
A novel nanocomposite based on fluorescent turn-on gold nanostars for near-infrared photothermal therapy and self-theranostic caspase-3 imaging of glioblastoma tumor cell
Authors: Wang, J., Zhou, Z., Zhang, F., Xu, H., Chen, W., Jiang, T.
Journal: Colloids Surf B Biointerfaces (2018): 303-311
Cell-permeable organic fluorescent probes for live-cell long-term super-resolution imaging reveal lysosome-mitochondrion interactions
Authors: Han, Y., Li, M., Qiu, F., Zhang, M., Zhang, Y. H.
Journal: Nat Commun (2017): 1307
Field-Controlled Charge Separation in a Conductive Matrix at the Single-Molecule Level: Toward Controlling Single-Molecule Fluorescence Intermittency
Authors: Kennes, K., Dedecker, P., Hutchison, J. A., Fron, E., Uji, I. H., Hofkens, J., Van der Auweraer, M.
Journal: ACS Omega (2016): 1383-1392
Determination of equilibrium and rate constants for complex formation by fluorescence correlation spectroscopy supplemented by dynamic light scattering and Taylor dispersion analysis
Authors: Zhang, X., Poniewierski, A., Jelinska, A., Zagozdzon, A., Wisniewska, A., Hou, S., Holyst, R.
Journal: Soft Matter (2016): 8186-8194

References

View all 1 references: Citation Explorer
Quantitative comparison of long-wavelength Alexa Fluor dyes to Cy dyes: fluorescence of the dyes and their bioconjugates
Authors: Berlier JE, Rothe A, Buller G, Bradford J, Gray DR, Filanoski BJ, Telford WG, Yue S, Liu J, Cheung CY, Chang W, Hirsch JD, Beechem JM, Haugl and RP., undefined
Journal: J Histochem Cytochem (2003): 1699
Page updated on November 3, 2024

Ordering information

Price
Unit size
Catalog Number2856
Quantity
Add to cart

Additional ordering information

Telephone1-800-990-8053
Fax1-800-609-2943
Emailsales@aatbio.com
InternationalSee distributors
Bulk requestInquire
Custom sizeInquire
Technical SupportContact us
Purchase orderSend to sales@aatbio.com
ShippingStandard overnight for United States, inquire for international
Request quotation

Physical properties

Molecular weight

843.42

Solvent

DMSO

Spectral properties

Correction Factor (260 nm)

0.06

Correction Factor (280 nm)

0.05

Extinction coefficient (cm -1 M -1)

150000

Excitation (nm)

645

Emission (nm)

663

Quantum yield

0.651

Storage, safety and handling

Certificate of OriginDownload PDF
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22

Storage

Freeze (< -15 °C); Minimize light exposure
UNSPSC12352200
Product Image
Product Image
Gallery Image 1
Fluorescent ATTO dye NHS esters (or succinimidyl esters) are the most popular tool for conjugating ATTO dyes to a peptide, protein, antibody, amino-modified oligonucleotide or nucleic acid. NHS esters react readily with the primary amines (R-NH<sub>2</sub>) of proteins, amine-modified oligonucleotides, and other amine-containing molecules. The resulting dye conjugates are quite stable.